Propolis extract

ABSTRACT

Disclosed are a propolis extract where the peculiar odor of propolis is reduced by decreasing the odorizing ingredients, a process to produce the propolis extract which comprises soaking propolis in a hydrophilic organic solvent to effect extraction and treating the liquid layer in the resultant extract with an anion-exchange resin, and a biologically active composition comprising the propolis extract.

BACKGROUND OF THE INVENTION

1. Field of the Invention

This invention relates to a novel propolis extract, in particular, to apropolis extract where the peculiar odor of propolis is reduced.

2. Description of the Prior Art

Propolis is known as a sticky substance which is used to build thebeehives: It is said that there are present in intact propolis a varietyof trace ingredients in form of a homogenous mixture with resins,beeswax, essential oils and pollens as predominant ingredients. Propolishas been used from ancient times as medicine for folk remedy indifferent areas in the world because of its potential antimicrobial andlenitive activities. As described in Shinobu Matsuda, "Foods and FoodIngredients Journal of Japan", No.160, pp.64-73 (1994), recently, otheringredients such as flavonoids and phenol carboxylic acids have beenisolated and identified one after another. With the advances inidentification and clarification of particular ingredients and theirbiological activities, the usefulness of propolis as life improvingmeans has been highlighted, thus a variety of health-care andsupplemental health-care products have appeared in the market.

By the way, intact propolis is not suitable for use in food productsbecause it usually contains large amounts of contaminants and hardlydissolves in water. In commercialized health-care and supplementalhealth-care products, propolis is added in the form of extracts whichare obtained through the step of soaking crushed propolis mass inethanol, glycerol and/or water. Propolis has a peculiar odor, which isone of the drawbacks to be overcome but however still not overcome incurrently available propolis extracts, thus restricting their use to afew types of food products. Although Japanese Patent Kokai Nos.165,595/89 and 316,968/93 propose several approaches to improve theappearance and smell of propolis extracts using activated charcoals,synthetic porous adsorbents and cation-exchange resins, each of theapproaches has been proved unsuccessful in reducing the peculiar odor ofpropolis.

SUMMARY OF THE INVENTION

In view of the foregoing, the first object of this invention is toprovide a propolis extract where the peculiar odor of propolis isreduced.

The second object of this invention is to provide a process to producesuch a propolis extract.

The third object of this invention is to provide several uses of such apropolis extract.

The present inventors investigated the odorizing ingredients in propolisusing gas chromatography and sensory test in combination, resulting inthe finding that on gas chromatography using p-ethyl phenol as internalstandard, one major odorizing ingredient gives a relative retention timeagainst p-ethyl phenol of 0.7-0.8, and another major odorizingingredient, a relative retention time against p-ethyl phenol of 1.2-1.3.The present inventors further investigated various procedures which mayremove such ingredients, eventually finding that the ingredients can besuccessfully removed by soaking propolis in hydrophilic organic solventsto effect extraction, and treating the liquid layer in the resultantextract with anion-exchange resins: Thus the peculiar odor of propoliscan be reduced without attenuating the biological activities inherent topropolis. Further, the present inventors confirmed that the propolisextract thus obtained is favorably feasible in various uses which havebeen never realized with conventional propolis extracts because of thepeculiar odor of propolis, in addition to the same uses as inconventional propolis extracts, thus completing this invention.

More particularly, to attain the first object, this invention provides apropolis extract where the content of an odorizing ingredient with arelative retention time against p-ethyl phenol of 0.7-0.8 as determinedon gas chromatography using p-ethyl phenol as internal standard, and/oranother odorizing ingredient with a relative retention time of 1.2-1.3determined similarly as above is or are reduced.

To attain the second object, this invention provides a process toproduce such a propolis extract, comprising soaking propolis in ahydrophilic organic solvent to effect extraction, and treating theliquid layer in the resultant extract with an anion-exchange resin.

To attain the third object, this invention provides a biologicallyactive composition which contains such a propolis extract.

BRIEF EXPLANATION OF THE ACCOMPANYING DRAWINGS

FIG. 1 is the gas chromatogram of a propolis extract which has beentreated with an anion-exchange resin.

FIG. 2 is the gas chromatogram of another propolis extract which has notbeen treated with anion-exchange resin.

In FIGS. 1 and 2, the symbol "A" indicates the peak of the odorizingingredient A; "B", the odorizing ingredient B; and "C", the internalstandard p-ethyl phenol.

DETAILED DESCRIPTION OF THE INVENTION

This invention is based on the identification of odorizing ingredientsin propolis and discovery of means which enable successful removal ofthe ingredients. As described heretofore, major odorizing ingredients inpropolis give a relative retention time against p-ethyl phenol of either0.7-0.8 or 1.2-1.3 on gas chromatography using p-ethyl phenol asinternal standard. Conventional propolis extracts contain the formerodorizing ingredient in an amount not less than 4 mg/g of solid, and thelatter odorizing ingredient in an amount not less than 60 mg/g of solid,dependently on the source of propolis as starting material and way ofextraction. Further, the results of sensory test confirmed that thepeculiar odor of propolis is reduced when the content of the formerand/or latter odorizing ingredient(s) is or are reduced, as well as thatthe peculiar odor become substantially not to be perceived when theformer and latter odorizing ingredients are less than 2.5 mg/g-solid and40 mg/g-solid respectively. Thus, the wording "propolis extract of thisinvention" as referred to in this invention includes propolis extractsin general regardless of their nature and source of propolis as startingmaterial as well as of way of preparation, as long as their content ofan odorizing ingredient having a relative retention time against p-ethylphenol of 0.7-0.8 as determined on gas chromatography using p-ethylphenol as internal standard, and/or another odorizing ingredient with arelative retention time against p-ethyl phenol of 1.2-1.3 determinedsimilarly as above is or are reduced.

Such a propolis extract can be prepared in desired amounts by theprocess according to this invention, comprising the step of extractionusing hydrophilic organic solvents, and treatment with anion-exchangeresins. In the process according to this invention, propolis is firstsoaked in a hydrophilic organic solvent to effect extraction, then theliquid layer in the resultant extract is collected and treated with ananion-exchange resin to remove odorizing ingredients. As to the sourceof propolis as starting material, one can use those from Japan, SouthAmerica, North America, Australia, China and Europe: Propolis in massiveform may be crushed and/or pulverized prior to extraction in order toimprove extraction efficiency. The wording "hydrophilic organic solvent"as referred to in this invention includes any organic solvents, as longas they dissolve in water: Preferred hydrophilic solvents are, forexample, lower alcohols such as methanol, ethanol, n-propanol andisopropanol which are usually used in mixture with water.

As to extraction conditions, propolis is soaked in equivolume,preferably, 5-fold volume of either hydrophilic organic solvent or amixture of hydrophilic solvent and water, and extracted at a temperatureexceeding ambient temperature, usually, 40° C. or higher, preferably,50°-80° C. for 0.5 hours or longer, preferably, 1 hour or longer whilestirring. Usually, extraction is repeatedly conducted: For example, inthe case of using a mixture of hydrophilic organic solvent and water,the ratio of hydrophilic solvent is first set to a lower level, thenelevated stepwise in every repeated extraction. More particularly,propolis is soaked in such a mixture with a content of hydrophilicorganic solvent of 30% (v/v) or less, then the solid layer is collectedand soaked again in a mixture with a content for hydrophilic organicsolvent exceeding 30% (v/v) to effect further extraction. In this case,one can favorably conduct extraction when the counter current method isemployed in a continuous or semi-continuous manner. The propolis whichhas been subjected to such an extraction consists of a solid layer,which is rich in contaminants, and a liquid layer which is rich inbiologically active ingredients. The liquid layer is collected by, forexample, filtration, decantation and/or centrifugation, adjusted to adesired concentration, if necessary, and then transferred to thetreatment step using anion-exchange resin.

The wording "anion-exchange resin" as referred to in this inventionmeans strongly, intermediately and weakly basic anion-exchangers ingeneral, which comprise a polymer base of three-dimensionallypolymerized hydrocarbons in powder, granule, bead or hollow fiber form,and primary amino groups, secondary amino groups, tertiary amino groupsand/or quaternary ammonium groups as ion exchange groups bound on thepolymer base. Commercially available anion-exchange resins are, forexample, "DIAION WA-10", "DIAION WA-11", "DIAION WA-20", "DIAION WA-21"and "DIAION WA-30" (products of Mitsubishi Chemical Corp., Tokyo,Japan), "AMBERLITE IRA411S", "AMBERLITE RA458" and "DUOLITE A-30B"(products of Rohm and Haas Co., Philadelphia, Pa., U.S.A.), and "A21"and "A26" (products of Japan Organo Co., Ltd., Tokyo, Japan), which maybe used in combination with cation-exchange resins and/or syntheticporous adsorbent, if necessary. One can easily obtain high-qualitypropolis extracts when anion-exchange resins are used in combinationwith cation-exchange resins.

The liquid layer can be treated batchwise or in continuous manner: Whenthe liquid layer to be treated is in large amount, it is much morefavorable to employ a continuous operation where anion-exchange resin ispacked in column inside a cylindrical tube of glass, ceramic orstainless steal. In each case, anion-exchange resin is used at 1% ormore, preferably, 5% or more in wet weight against the amount of solutesin liquid layer to be treated, and the treating conditions areadequately modulated so that the liquid layer is kept in contact withanion-exchange resin over a time period of 0.1 hour or longer,preferably, 0.5 hours or longer. For example, in the case of usinganion-exchange resin packed in column, a mixture of hydrophilic organicsolvent and water is applied through the column for its equilibration,and the liquid layer is loaded through one end of the column, followedby applying a fresh preparation of the same mixture as described aboveat SV5 or less, preferably, SV3 or less. The eluate from the column canbe fractionated, and the fractions with biologically active ingredientsare collected, pooled and concentrated followed by dehydrating intosolution, syrup or solid, if necessary. In the propolis extract obtainedaccording to this invention, usually, the peculiar odor of propolis issubstantially eliminated and this may render the extract odorless.

The propolis extract of this invention bears the biological activitiesinherent to propolis, for example, antimicrobial activity, antiviralactivity, antitumor activity, anti-inflammatory activity,anti-hyperlipemic activity, local anesthesia activity, immune-regulatoryactivity, apoptosis-regulatory activity, anti-oxidative activity, ultraviolet ray-absorbing activity, preservative activity and vitaminP-supplying activity. The propolis extract of this invention isfavorably usable alone or in combination with other biologically activecompositions in the fields of foods, cosmetics (including toiletries)and pharmaceuticals, as well as in other fields where the biologicalactivities of propolis are needed. Thus, the wording "biologicallyactive composition" as referred to in this invention incudes anycompositions regardless of their form, as long as they are formulatedwith the propolis extract of this invention and feasible in the fieldsas described above where the biological activities of propolis areneeded and used. Such a composition is usually provided in forms whichare feasible in the above described field without further processing, aswell as in the form of starting materials and ingredients which are usedin the preparation of final products and, further, in the form ofintermediates which are added in any stages of preparing final products.

The use of the propolis extract of this invention in particular fieldswill be further explained in detail: In the field of foods, the presentpropolis extract can be favorably used alone as supplemented heath-careproduct or together with other one or more materials and/or ingredientswhich are usually used in food products, for example, water, alcohols,starches, proteins, dietary fibers, saccharides, lipids, vitamins,minerals, flavorings, colorings, sweeteners, seasonings, spices,emulsifiers, antiseptics, preservatives, fungicides, germicides andantioxidants, to facilitate the intake of the propolis extract of thisinvention. The biologically active composition according to thisinvention is prepared into solution, emulsion, syrup, cream, paste,jelly, powder, granule and other desired solid forms to meet toparticular final uses. The biologically active composition directed touse in the field of foods usually contains the present propolis extractin an amount of 0.001% (w/w) or more, preferably, 0.005% (w/w) or moreon a dry solid basis (d.s.b.).

Particular forms in foods are, for example, seasonings such as a soysauce, powdered soy sauce, "miso", "funmatsu-miso" (a powdered miso),"moromi" (a non-refined sake), "hishio" (a non-refined soy sauce),"furikake" (a seasoned fish meal), mayonnaise, dressing, vinegar,"sanbai-zu" (a sauce of sugar, soy sauce and vinegar),"funmatsu-sushi-su" (powdered vinegar for sushi), "tentsuyu" (a saucefor Japanese deep-fat fried food), "mentsuyu" (a sauce for Japanesevermicelli), sauce, catsup, "yakiniku-no-tare" (a sauce for Japanesegrilled meat), curry roux, "chuka-no-moto" (a seasoning for Chinesedish), instant stew mix, instant soup mix, "dashi-no-moto" (an instantstock mix), mixed seasoning, "mirin" (a sweet sake), "shin-mirin" (asynthetic mirin), table sugar, and coffee sugar; "wagashi" (Japanesecakes) such as a "senbei" (a rice cracker), "arare" (a baked rice cake),"okoshi" (a millet-and-rice cake), "karinto" (fried dough cake), "gyuhi"(starch paste), "mochi" (a rice cake), "manju" (a bun with a bean-jam),"uiro" (a sweet rice jelly), "an" (a bean jam), "yokan" (a sweet jellyof beans), "mizu-yokan" (a soft adzuki-bean jelly), "kingyoku" (a kindof yokan), jelly, pao de Castella, and "amedama" (a Japanese toffee);confectioneries such as a biscuit, cracker, cookie, pie, pudding, creampuff, waffle, sponge cake, doughnut, chocolate, chewing gum, caramel,candy, and gummy jelly; frozen desserts such as an ice cream, ice candy,and sherbet; syrups such as a "korimitsu" (a sugar syrup for shavedice); spreads and pastes such as a butter cream and custard cream;processed fruits and vegetables such as a jam, marmalade, "syrup-zuke"(a fruit pickle with a syrup), and "toka" (a conserve); processed cerealfoods such as a bun, noodle, cocked rice, and artificial meat; oil andfat foods such as a salad oil and margarine; pickles or pickled productssuch as a "fukujin-zuke" (red colored radish pickles), "bettara-zuke" (akind of whole fresh radish pickles), "senmai-zuke" (a kind of slicedfresh radish pickles) and "rakkyo-zuke" (pickled shallots); premixes forpickles or pickled products such as a "takuan-zuke-no-moto" (a premixfor pickled radish), and "hakusai-zuke-no-moto" (a premix for freshwhite rape pickles); meat products such as a ham and sausage; fishproducts such as a fish ham, fish sausage, "kamaboko" (a steamed fishpaste), "chikuwa" (a kind of fish paste), and "hanpen" (a Japanesedeep-fat fried fish paste); "chinmi" (relishes) such as an"uni-no-shiokara" (salted guts of sea urchin), "ika-no-shiokara" (saltedguts of squid), "su-konbu" (a processed tangle), "saki-surume" (driedsquid strips), and "fugu-no-mirin-boshi" (a dried mirin-seasonedswellfish); boiled foods such as those cooked with agriculturalproducts, livestocks, and fisheries; daily dishes such as a boiled food,grilled food, fry, fried food, steamed food, and dishes dressed withsauce; frozen foods such as those of shrimp for frying, croquette,shao-mai, "gyoza" (fried or steamed dumpling stuffed with minced pork),"harumaki" (a kind of Chinese dish), hamburger steak, meat ball, fishhamburger, or fish ball; retort foods such as those of a hamburger, meatball, rice boiled together with red beans, rice boiled with beef orchicken, gruel of unpolished rice, curry, meat sauce, demiglace sauce,potage soup, consomme soup, stew, Japanese hotchpotch, "happosai" (akind of Chinese vegetable dish with meats and fish), boiled bean,grilled chicken, pot-steamed hotchpotch, boiled chestnut, andwater-boiled vegetable; egg and milk products such as a "kinshi-tamago"(a stripped egg roll), milk beverage, butter, and cheese; canned andbottled products such as those with meats, fish meats, fruits, orvegetables; alcohols such as a synthetic sake, sake, wine, and liquor;soft drinks such as a coffee, cocoa, juice, green tea, black tea, Oolongtea, mineral beverage, carbonated beverage, sour milk beverage, andbeverage containing lactic acid bacteria; and instant food products suchas an instant pudding mix, instant hot cake mix, instant juice,"sokuseki-shiruco" (an instant mix of adzuki-bean soup with rice cake),and instant soup mix. In addition to foods for humans, the presentpropolis extract can be also used in feeds and pet foods for animalssuch as domestic animals, poultry, honey bees, silk worms, and fishes.

In the field of cosmetics, including toiletries, the present propolisextract can be, while used alone, favorably used together with one ormore commonly feasible ingredients for cosmetics to facilitate theapplication of the extract. Examples of the feasible ingredients areoily bases, aqueous bases, -flavorings, colorings, dyes, refrigerants,humectants, emollients, emulsifiers, gelatinizers, viscosity enhancers,softening agents, solubilizers, surfactants, foam-stabilizers,stabilizers, clearances, adipositas agents, putrefactive agents,coating-forming agents, propellants, antisepses, preservatives, andantioxidants. The composition can be incorporated further together withone or more agents such as vitamins, amino acids, peptides, hormones,extracts, vasodilators, blood circulation-improving agents,cell-activators, germicides, anti-inflammatory drugs,urtication-preventing agents, astringents, skin-function-promotingagents, and keratolytics. The composition is prepared into solution,emulsion, cream, paste, powder, granule and other desired solid forms toconform to the particular final uses. The biologically activecomposition directed to use in the field of cosmetics usually containsthe present propolis extract in an amount of 0.001% (w/w) or more,preferably, 0.005% (w/w) or more (d.s.b.).

Particular examples of the present biologically active composition inthe form of cosmetics are those for hair such as hair restorers and hairgrowth-promoting agents, pomades, hair sticks, hair oils, hair creams,hair solids, hair liquids, hair set lotions, hair styling-gels, hairwater-greases, hair mousses, hair aerosols, hair liquids for permanentwave, and hair dyes; those for washing such as shampoos for hair andbody, hair rinses, hair-washing soaps, cosmetic soaps, and settingfoams; those for skins such as cosmetic water, creams, milky lotions,lotions, packs, foundations, lip sticks, rouges, eye liners, mascara,eye shadows, eyebrow pencils, manicures, and powders; those for oraluses such as tooth powders, moisturized dentifrices, toothpastes, toothwashes, medical dentifrices, cachous, and gargles; and other cosmeticssuch as sunscreens, shaving cosmetics, bath cosmetics, perfumes, eau decolognes, underarm deodorants, baby powders, eye lotions, and bleachingcreams. In the cosmetics for skins or hair, the present propolis extractmore readily functions when co-incorporated with α-glycosylbioflavonoids such as α-glucosyl rutin, α-glucosyl hesperidin, andα-glucosyl naringin in a final amount of about 0.001% (w/w) to about 10%(w/w), because the bioflavonoids promote the supplement of nutrients toskins and the metabolism of living bodies. The present propolis extractalso more readily functions when co-incorporated with humectants such asmoisture-retaining saccharides or sugar alcohols, for example, maltose,trehalose, and maltitol at an appropriate content, preferably, in afinal amount of 1% (w/w) or lower, because the humectants keep theskins, scalps, and/or hair well moistened.

In the field of pharmaceuticals, the present propolis extract can beused in pharmaceutical compositions to treat and/or prevent the presentextract-susceptive diseases such as infections, autoimmune diseases,malignant tumors, and hyperlipemias, which may emerge in the alimentarysystem, circulating system, urinary and genital organs, brain andnervous system, eye, ear, nose, throat, or skin.

In the field of pharmaceuticals, an effective amount of the presentpropolis extract can be used alone or, if necessary, together with oneor more conventional agents for pharmaceuticals. Examples of theconventional agents are anesthetics, hypnotic sedatives, anti-anxieties,antiepileptics, antipyretic antiphlogistics, stimulants, wake amines,anti-parkinson drugs, agents for psychoneuroses, agents for centralnervous system, skeletal muscle relaxants, agents for autonomic nervoussystem, antispastic agents, drugs for eye, drugs for nose and ear,anti-vertiginous drugs, cardiotonics, antiarrhythmic drugs, diuretics,pressure reduction drugs, vasoconstrictors, coronary vaso-dilators,peripheral vasodilating drugs, hyperlipemia drugs, breath stimulants,antitussive and expectorant drugs, bronchodilators, drugs for allergy,antidiarrheal drugs, drugs for intestinal disorders, peptic ulcer drugs,stomachic digestants, antacids, cholagogouses, pituitary hormone drugs,salivary gland hormones, thyroid hormone drugs, antithyroid drugs,anabolic steroids, corticosteroids, androgen drugs, estrogen drugs,corpus luteum hormone drugs, mixed hormones, urinary/genital organdrugs, anus drugs, surgical sterilizations/antiseptics, woundprotectives, externals for purulent diseases, analgesics, antipruritics,astringents, antiphlogistics, externals for parasitic skin diseases,skin-softening drugs, caustics, dental/oral drugs, vitamins, inorganicpreparations, supplemental liquids, hemostatics, anticoagulation drugs,drugs for liver diseases, antidotes, habitual intoxication drugs, drugsfor treatment of gout, enzyme preparations, diabetic drugs,antioncotics, antihistaminics, drugs for stimulation treatment,antibiotics, chemotherapeutics, biological preparations, anthelmintics,anti-Protozoas, drugs for preparations, X-ray contrast media, anddiagnostic agents. The composition can be incorporated further togetherwith one or more ingredients to facilitate the ingestion of the presentpropolis extract as a medical agent. Examples of the ingredients areadjuvants, diluents, bodies, stabilizers, carriers, colorings,flavorings, antiseptics, and preservatives. The resultant compositioncan be desirably formed for application. Examples of the forms areextracts, elixirs, capsules, granules, pills, ointments for eye,suspensions, emulsions, plasters, suppositories, powders, alcoholpreparations, tablets, syrups, infusions, feeding fluid, decoctions,injections, tinctures, ophthalmic solutions, troches, ointments,cataplasms, aromatic water, liniments, lemonades, fluidextracts,lotions, nasal drops, nasal nebulas, inhalants for lower airway,sustained release drugs for eye, oral mucosal patches, and enemas. Adosage per day of the present propolis extract for pharmaceutical use isselected dependently on the aims and administration routes, usually from0.01 mg to 100 mg for an adult human. The present biologically activecomposition, containing the present propolis extract which is a naturalproduct with biological activities, does not induce harmful side effectsto mammals and humans even in daily uses. Therefore healthy humans alsocan use present composition in daily lives, without anxieties, toprevent the susceptive diseases and improve living activities.

The biologically active composition of this invention can be also usedas, for example, an antiseptic and antioxidant, because the compositionbears the biological activities inherent in propolis. The presentcomposition for such uses can be used in the fields, including those offoods, cosmetics, and pharmaceuticals, that must suppress proliferationof microbes and oxidization of effective ingredients. As an antisepticand antioxidant, the present propolis extract can be used alone ortogether with one or more commonly feasible ingredients in respectivefields. Examples of the feasible ingredients are antiseptics andantioxidants other than propolis extracts, preservatives, solvents,solubilizers, viscocitizers, and flavorings. Depending on the purposes,the composition is prepared into solution, suspension, syrup, paste,powder, granule and other desired solid forms to meet the particularfinal uses. The composition can be used as a starting or intermediatematerial to be added to foods, cosmetics, pharmaceuticals, germicidalagents, and antimicrobial agents; effecting antisepsis, antioxidation,preservation, germicide, antimicrobe, etc., in the final products. Thecomposition can be also used by itself as an antiseptic or antioxidantwhich is used by spraying or coating to daily utensils. The presentcomposition directed to use as an antiseptic or antioxidant contains thepresent propolis extract in an amount of 0.01% (w/w) or more,preferably, 0.1% (w/w) or more (d.s.b.).

The present propolis extract can exhibit remarkable antimicrobialactivity against fungi, yeasts, bacteria, algae, etc. The extract istherefore useful in a germicidal or antimicrobial agent which is appliednot only to humans directly but also to daily articles. For such use,the present propolis extract is usually mixed with a desirable solventor diluent and additional ingredients selected depending on the uses.Examples of the additional ingredients are metals such as silver, saltssuch as zeolite (a type of aluminosilicate), flavorings, colorings,propellants, surfactants, binder resins, pH modulators, and buffers. Theresultant mixtures can be provided after being impregnated in or appliedto a paper, film, sheet, cloth, non-woven fabric, hard foam rubber,semi-soft foam rubber, or soft foam rubber, or packed in tubes, spraycans, or bottles.

The germicidal agent and antimicrobial agent according to this inventioncan be used similarly as conventional ones, for example, by wholly orpartly spraying or coating to articles susceptible to microbialcontamination. Examples of the articles to be coated or sprayed arepersonal articles including clothes, caps, hats, shoes, blades, hairsetting or styling utensils, toothbrushes, watches, glasses, bags,purses, umbrellas, and parasols; writing utensils including pens andpencils; household supplies including beds, bed clothes, airconditioners and filters thereof, cleaners, humidifiers, remotecontrollers, toys, sinks, toilet bowls, doorknobs, toilet papers, bathtubs, curtains, shower curtains, carpets, rugs, children's chairs,partitions for children, and waxes; kitchen utensils includingrefrigerators, can openers, toasters, cooking utensils, juicers, cookingknives, pans, mixers, weights, sieves, strainers, sponges, and lunchboxes; daily necessaries including soaps, detergents, wet towels, wettissue papers, towels, and mats; building materials including adhesives,caulking agents, concretes, cements, mastics, and paints; communicationmedia including telephones, calculators, office computers, cameras,video cameras, rental video tapes, rental records, and rental books;games including mahjongg tiles, playing cards, chessboards and chessmen(including Japanese style, i.e., "Shogiban" and "Shogi-no-koma"), andcheckerboards and checkermen (including Japanese style, i.e., "Goban"and "Goishi"); athletic utensils or instruments, musical instruments,sanitary napkins and tampons, credit cards, and handles and steeringwheels of cars. The present germicidal agent and antimicrobial agent canbe used further together with other antiseptics and, if possible,incorporated in the articles as mentioned above during their productionprocesses by the step of mixing, kneading, dissolving, injecting, orsoaking.

The addition of organic acids to the present propolis extract canenhance one or more of the biological activities thereof. The wording"organic acid" as referred to in this invention includes any organicacid, including its derivatives such as salts, that can be incorporatedin foods, cosmetics, pharmaceuticals, germicidal agents, orantimicrobial agents. Examples of the organic acids are citric acid,lactic acid, DL-tartaric acid, L-tartaric acid, acetic acid, butyricacid, dehydrobutyric acid, adipic acid, propionic acid, fumaric acid,malic acid, L-aspartic acid, L-glutamic acid, cinnamic acid, L-ascorbicacid, nicotinic acid, folic acid, gluconic acid, benzoic acid, sorbicacid, boric acid, trisodium citrate, monopotassium citrate, sodiumlactate, calcium lactate, sodium DL-tartrate, potassium L-tartrate,sodium L-tartrate, sodium acetate, calcium propionate, sodiumpropionate, monosodium fumarate, sodium L-aspartate, calciumL-glutamate, sodium dehydroacetate, sodium benzoate, and potassiumsorbate. These examples can be used in combination, if necessary. To thepresent propolis extract, the organic acids in a solid form can be addedat a ratio of 1/2-fold or more, preferably one to 40 times, on a drysolid basis; and in a liquid form, 1/2-fold or more, preferably one to40 times, by liquid weight to the total dry solid weight of the extract.The most preferable organic acid is citric acid. For example, thepresent propolis extract mixed with five times its weight of citric acidexhibits antimicrobial activity with a synergistically enhancedintensity, apparently higher than the intensity estimated from the sumof the respective activities. The biologically active composition ofthis invention which further contains the organic acids is feasible inany field as mentioned above.

The present biologically active composition can be prepared in the formof a composition with, as mentioned above, an antiseptic other thanpropolis extracts. The wording "antiseptic other than propolis extracts"as referred to in this invention includes any cytostatic or cytocidalcompounds against a fungus, yeast, bacterium, or alga that can beincorporated in foods, cosmetics, pharmaceuticals, germicidal agents, orantimicrobial agents. Examples of the antiseptics other than propolisextracts are ε-polylysine, butyl parahydroxybenzoate, propylparahydroxybenzoate, biphenyl hydroxybenzoate, benzoic acid, sodiumbenzoate, sorbic acid, and potassium sorbate, which may be alsoclassified with the above-mentioned organic acid. These examples can beused in combination, if necessary. More preferably, the antisepticseffective against fungi are used in this invention. To the presentpropolis extract, the antiseptics in a solid form can be added at aratio of 1/2-fold or more, preferably one to 20 times, on a dry solidbasis; and in a liquid form, 1/2-fold or more, preferably one to 20times, by liquid weight to the total dry solid weight of the extract.The biologically active composition of this invention which furthercontains the antiseptics other than propolis extracts is feasible in anyfield as mentioned above. The antiseptic ε-polylysine is a naturalcompound which is effective against fungi and acceptable forincorporation in foods. The present biologically active compositioncontaining ε-polylysine is therefore safely and advantageously used infoods.

The following experiments explain the properties and biologicalactivities of the present propolis extract.

Experiment 1

Preparation of Propolis Extract

One kilogram of Brazilian propolis blocks were, after being crushed intopowders, soaked in 5 kg of 30% (v/v) aqueous ethanol at 60° C. for twohours, and then cooled to ambient temperature. The solid layer in theresultant soak was collected by filtration through kieselguhr and filterpaper, and soaked in 5 kg of 70% (v/v) aqueous ethanol at 60° C. for twohours for extraction. After being cooled to ambient temperature andbeing allowed to stand at 4° C. overnight, the liquid layer in theresultant extract was collected by filtration similarly as above. Theseparated solid layer was soaked as above for re-extraction, and newlyformed liquid layer in the resultant of re-extraction was collected byfiltration similarly as above and pooled with the above-obtained liquidlayer.

To a portion of the pooled liquid layer, 10% (w/w) weight (on a wetweight basis) of any one of the ion-exchange resins shown in Table 1which were pre-equilibrated with 70% (v/v) aqueous ethanol was added,and the liquid layer was moderately stirred at ambient temperature forone hour. The resultant mixture was centrifuged to collect thesupernatant, which was then concentrated and dehydrated in vacuo into asolid of a propolis extract. In the case of using an anion-exchangeresin, the resin was used in OH form; and a cation-exchange resin, Hform. For combined use of anion- and cation-exchange resins, the mixtureof the resins was used at a ratio of one to two on a wet weight basis.For control, another portion of the pooled liquid layer was concentratedand dehydrated into a solid without ion-exchange resin treatment. Thusseven kinds of propolis extract samples in solid form, numbered 1-7,were prepared for testing by the following experiments.

                  TABLE 1                                                         ______________________________________                                        Treatment                                                                     Anion-Exchange Resin                                                                       Cation-Exchange Resin                                                                        Sample Numbering                                  ______________________________________                                        AMBERLITE IRA411S                                                                          --             1                                                 (strongly basic)                                                              DUOLITE A-30B                                                                              --             2                                                 (intermediately basic)                                                        DIAION WA-30 --             3                                                 (weakly basic)                                                                DIAION WA-30 SK1B           4                                                 (weakly basic)                                                                             (strongly acidic)                                                AMBERLITE IRA411S                                                                          SK1B           5                                                 (strongly basic)                                                                           (strongly acidic)                                                --           SK1B           6                                                              (strongly acidic)                                                --           --             7                                                 ______________________________________                                    

Experiment 2

Sensory Test

The seven samples of Experiment 1 were subjected to a test for thepeculiar odor of propolis in usual manner by six panels who had adequateexperiences of smelling propolis. The results are summarized in Table 2,on the basis of the panels' judgements, with the following four ranks ofsensory evaluation on the peculiar odor of propolis: "+++", intense;"++", relatively intense; "+", relatively moderate; and "±", weak oralmost none.

                  TABLE 2                                                         ______________________________________                                        Sample No.  Sensory Evaluation                                                                         Note                                                 ______________________________________                                        1           +            This Invention                                       2           ±         This Invention                                       3           ±         This Invention                                       4           ±         This Invention                                       5           ±         This Invention                                       6           ++           Reference                                            7           +++          Control                                              ______________________________________                                    

As shown in Table 2, the peculiar odor of propolis was remarkablyreduced in the samples 1 to 5 as compared to the sample 7 for control.

Experiment 3

Identification of Odorizing Ingredient

The seven samples of Experiment 1 were analyzed by gas chromatographyusing p-ethyl phenol as internal standard as follows. A sample wasprecisely weighed out for 250 mg and dissolved in 5 ml of methanol. Tothe solution, 0.25 ml of 10,000 ppm p-ethyl phenol of methanol solutionwas added, and 2 μl of the resultant mixture was analyzed. The columnused was a capillary column chemically bound with polyethyleneglycolnitroterephthalate as liquid phase, "TC-FFAP" (0.53 mm in internaldiameter and 30 m in length, commercialized by GL Sciences Inc., Tokyo,Japan); and the chromatograph, "GC-17B" (commercialized by ShimadzuCorp., Kyoto, Japan). Helium gas was used as carrier gas at a flow rateof 10 ml/min. The temperature of sample injection gate was set at 250°C. The temperature in the column was programmed to remain at 40° C. forfive minutes after sample injection, rise at a rate of 5° C./min, andthen remain at 230° C. The detection was with a hydrogen flameionization detector. FIGS. 1 and 2 show, as typical results, the gaschromatograms of the samples 3 and 7, respectively.

The ingredients separated by gas chromatography as mentioned above werenext by a sensory test on odor according to "GC Sniff method", describedin Koji Wada et al., "Agricultural and Biological Chemistry", Vol.51,No.7, pp.1753-1760 (1987). In this test, gas released from the detectorwas directly smelled after hydrogen flow to the detector was suspendedand the flame stifled. Then, the above-obtained gas chromatograms werestudied by referring to the results of this sensory test, resulting inthe identification of two major odorizing or odoriferous ingredients: ongas chromatography using p-ethyl phenol as internal standard, one givesa relative retention time of 0.7-0.8 against p-ethyl phenol; and theother, a relative retention time of 1.2-1.3 against p-ethyl phenol. Theingredients were designated "A" and "B", respectively. On the basis ofthe peak areas in the chromatograms, the contents of the ingredients Aand B were calculated for the samples 1-7. The results are in Table 3.

                  TABLE 3                                                         ______________________________________                                                 Contents (mg/g)                                                      Sample No.                                                                              Ingredient A                                                                              Ingredient B                                                                            Note                                          ______________________________________                                        1         2.35        33.2      This Invention                                2         2.07        27.7      This Invention                                3         1.66        36.2      This Invention                                4         1.80        24.5      This Invention                                5         2.09        16.9      This Invention                                6         3.37        27.9      Reference                                     7         4.49        66.5      Control                                       ______________________________________                                    

As shown in Table 3, the sample 7 for control, which intensely exhibitedthe peculiar odor of propolis, contained 4.49 mg of the ingredient A and66.5 mg of the ingredient B per gram by dry weight. On the contrary, thesamples 1-5 exhibited apparently-reduced peculiar odor and containedless of both ingredients, at the respective amounts less than 2.5 mg andless than 40 mg per gram by dry weight. The strongly basicanion-exchange resin "AMBERLITE IRA411S" (commercialized by Rohm andHaas Co., Philadelphia, Pa., U.S.A.), employed for the sample 1, wasrelatively less efficient in reducing the odorizing ingredients,particularly in reducing the ingredient A, but the resin efficientlydecreased both ingredients when used in combination with the stronglyacidic cation exchange resin as shown by the results of the sample 5.The results of the sample 6 indicate that the cation exchange resin isless efficient in reducing the odorizing ingredients when used alone.

Experiment 4

Antimicrobial Activity

The samples 1-7 of Experiment 1 were tested for minimal inhibitoryconcentrations (MICs) against the proliferation of microbes inaccordance with the method described in Japanese Society ofChemotherapy, "Chemotherapy", Vol.29, pp.76-79 (1987) and Vol.27,pp.559-560 (1979). For the test media, sensitivity disc agar plates wereprepared to contain any one of the samples 1-7 at concentrations of 500μg/ml and its serial 1/2 dilutions. The microbes in Table 4 were, afterbeing proliferated in the usual manner, streaked were on the media witha platinum loop and cultured at 27° C. for 24 hours. Thereafter themedia were macroscopically examined for colony formation in comparisonwith control media free of the samples to determine MICs. The resultsare in Table 4.

                                      TABLE 4                                     __________________________________________________________________________                     MIC (μg/ml)                                               Microbe (strain')                                                                              1" 2" 3" 4" 5" 6" 7"                                         __________________________________________________________________________    Microsporum gypseum (IFO 8231)                                                                 31.3                                                                             31.3                                                                             31.3                                                                             62.5                                                                             62.5                                                                             31.3                                                                             31.3                                       Bacillus cereus (IFO 466)                                                                      62.5                                                                             62.5                                                                             62.5                                                                             125                                                                              125                                                                              62.5                                                                             62.5                                       Pseudomonas aeruginosa (IFO 3453)                                                              125                                                                              125                                                                              125                                                                              250                                                                              250                                                                              125                                                                              125                                        Propionibacterium acnes (JCM 6425.sup.T)                                                       250                                                                              250                                                                              250                                                                              500                                                                              500                                                                              250                                                                              250                                        Corynebacterium equi (IFO 3730)                                                                62.5                                                                             62.5                                                                             62.5                                                                             125                                                                              125                                                                              62.5                                                                             62.5                                       __________________________________________________________________________     ': The strain of which depository number includes "IFO" is deposited in       The Institute for Fermentation, Osaka (Osaka city, Osaka, Japan); and         "JCM", in Japan Collection of Microorganisms (in The Institute of Physica     and Chemical Research, Wako city, Saitama, Japan).                            ": The numbers 1-7 are the sample numbers.                               

As shown in Table 4, the propolis extracts according to this invention(the samples 1-5) exhibited antimicrobial activity with nearlyequivalent efficiency to control (the sample 7) and reference (thesample 6). The microbes Microsporum gypseum, Bacillus cereus,Pseudomonas aerginosa, Propionibacterium acnes, and Corynebacteriumequi, employed in this experiment, would be responsible fordermatophytosis, sitotoxism, dermatitis, or diphtheria. The aboveresults, showing wide antimicrobial spectra of the present propolisextract, indicate the effectiveness of the present propolis extract inwide uses including foods, cosmetics, pharmaceuticals, germicidalagents, and antimicrobial agents.

Experiment 5

Toxicity Test

Any one of the samples 1-7 of Experiment 1 was administered toseven-week-old dd mice through peroral routes. No deaths were observedeven at the highest dose of 2.5 g/kg of body weight. These resultsindicate the safety of the propolis extracts of this invention forintake by mammals including humans.

The followings explain the preferred examples of this invention.

EXAMPLE 1 Propolis Extract

One kilogram of Brazilian propolis blocks were, after being crushed intopowders, soaked in 5 kg of 30% (v/v) aqueous ethanol at 60° C. for twohours, and then cooled to ambient temperature. The solid layer in theresultant soak was collected by filtration through kieselguhr and filterpaper, and soaked in 5 kg of 70% (v/v) aqueous ethanol at 60° C. for twohours for extraction. After being cooled to ambient temperature andallowed to stand at 4° C. overnight, the liquid layer in the resultantextract was collected by filtration similarly as above. The separatedsolid layer was soaked as above for re-extraction, and the newly formedliquid layer in the resultant re-extraction was collected by filtrationsimilarly as above and pooled with the above-obtained liquid layer. Thepooled liquid layer contained about 35% (w/w) dry solids of the materialpropolis material.

To the pooled liquid layer, 10% weight (on a wet weight basis) of theweakly basic anion-exchange resin "DIAION WA-30" (in OH form,commercialized by Mitsubishi Chemical Corp., Tokyo, Japan) which hadbeen equilibrated with 70% (v/v) aqueous ethanol were added, and theliquid layer was moderately stirred at ambient temperature for one hour.After being separated from the resin by filtration, the liquid layer wasconcentrated and dehydrated in vacuo into a solid of the propolisextract of this invention. This step with the ion-exchange resinrecovered about 75% (w/w) dry solids. By gas chromatography according toExperiment 1, the propolis extract was determined to contain about 1.5mg of the ingredient A and about 35 mg of the ingredient B per gram bydry weight. In the extract the peculiar odor of propolis is apparentlyreduced, while the extract retains the biological activities inherent inpropolis including antimicrobial activity, antiviral activity, antitumoractivity, anti-inflammatory activity, anti-hyperlipemic activity,anti-oxidant activity, ultraviolet ray-absorbing activity, preservativeactivity, and vitamin P-supplying activity. The extract is useful, byitself and in combination with other ingredients, in the fields offoods, cosmetics, and pharmaceuticals and other fields where thebiological activities of propolis are needed.

EXAMPLE 2 Propolis Extract

Liquid layer in resultant extract of propolis was prepared and pooled inaccordance with Example 1. To one weight part of the pooled liquidlayer, on a wet weight basis, 0.066 weight part of the strongly basicanion-exchange resin "A26" (in OH form, commercialized by Japan OrganoCo., Ltd., Tokyo, Japan) and 0.033 weight part of the strongly acidiccation-exchange resin "15WET" (in H form, commercialized by Japan OrganoCo., Ltd., Tokyo, Japan) both of which had been equilibrated with 70%(v/v) aqueous ethanol were added, and the liquid layer was moderatelystirred at ambient temperature for one hour. The liquid layer was then,in accordance with Example 1, separated from the resins, concentrated,and dehydrated in vacuo into a solid of the propolis extract of thisinvention. This step with the ion-exchange resins recovered about 70%(w/w) dry solids. By gas chromatography according to Experiment 3, thepropolis extract was determined to contain about 1.8 mg of theingredient A and about 20 mg of ingredient B per gram by dry weight. Inthe extract the peculiar odor of propolis is apparently reduced, whilethe extract retains the biological activities inherent in propolisincluding antimicrobial activity, antiviral activity, antitumoractivity, anti-inflammatory activity, anti-hyperlipemic activity,anti-oxidant activity, ultraviolet ray-absorbing activity, preservativeactivity, and vitamin P-supplying activity. The extract is useful, byitself and in combination with other ingredients, in the fields offoods, cosmetics, and pharmaceuticals and other fields where thebiological activities of propolis are needed.

EXAMPLE 3 Propolis Extract

The weakly basic anion-exchange resin "DIAION WA-30" (in OH form,commercialized by Mitsubishi Chemical Corp., Tokyo, Japan) and thestrongly acidic cation-exchange resin "SK1B" (in H form, commercializedby Mitsubishi Chemical Corp., Tokyo, Japan) were packed in a stainlesssteel column in a ratio of two to one by wet weight. After equilibrationof the column with 70% (v/v) aqueous ethanol, to the column a liquidlayer prepared and pooled by the method in Example 1 was applied, and70% (v/v) aqueous ethanol was run at a flow speed of SV3. The eluatefrom the column was collected, evaporated, and dehydrated in vacuo intoa solid of the propolis extract of this invention. This step recoveredabout 75% (w/w) dry solids. By gas chromatography according toExperiment 3, the propolis extract was determined to contain about 1.8mg of the ingredient A and about 25 mg of the ingredient B per gram bydry weight. In the extract the peculiar odor of propolis is apparentlyreduced, while the extract retains the inherent activities, includingantimicrobial activity, antiviral activity, antineoplastic activity,anti-inflammatory activity, anti-hyperlipemic activity, anti-oxidantactivity, ultraviolet absorbing activity, preserving activity, andvitamin P-supplying activity. The extract is useful, by itself and incombination with other ingredients, in the fields of foods, cosmetics,and pharmaceuticals and in other fields where the biological activitiesof propolis are needed.

EXAMPLE 4 Biologically Active Composition

A type of the biologically active composition of this invention wasprepared by dissolving one weight part of the propolis extract ofExample 1, four weight parts of citric acid, and three weight parts ofε-polylysine in 32 weight parts of 70% (v/v) aqueous ethanol. Thecomposition exhibits the biological activities inherent in propolis,particularly remarkable in anti-microbial activity, while the peculiarodor of propolis therein is reduced. The product is useful as a materialfor foods, cosmetics, and pharmaceuticals to maintain or promote health,and as an antiseptic, preservative, fungicide, and antioxidant feasiblein the products.

EXAMPLE 5 Biologically Active Composition

A type of the biologically active composition of this invention wasprepared by dissolving one weight part of the propolis extract ofExample 2 and five weight parts of acetic acid in 54 weight parts of 70%(v/v) aqueous ethanol. The composition exhibits the biologicalactivities inherent in propolis, particularly remarkable inantimicrobial activity, while the peculiar odor of propolis therein isreduced. The composition is useful as a material for foods, cosmetics,and pharmaceuticals to maintain or promote health, and as an antiseptic,preservative, fungicide, and antioxidant feasible in the products.

EXAMPLE 6 Biologically Active Composition

The propolis extract of Example 3 was crushed in the usual manner intopowders, and a type of the biologically active composition of thisinvention was prepared by mixing one weight part of the powders with twoweight parts of citric acid into homogeneity. The composition exhibitsthe biological activities inherent in propolis, particularly remarkablein antimicrobial activity, while the peculiar odor of propolis thereinis reduced. The composition is useful as a material for foods,cosmetics, and pharmaceuticals to maintain or promote health, and as anantiseptic, preservative, fungicide, and antioxidant feasible in theproducts.

EXAMPLE 7 Gummy Candy

One hundred and fifty weight parts of a hydrogenated maltooligosylsaccharide syrup, "MABIT®" (commercialized by Hayashibara Shoji, Inc.,Okayama, Japan), were concentrated by evaporation with heating to arelative moisture of about 15% (w/w). To the concentrate the followingmaterials were added: 13 weight parts of gelatine which had beendissolved in 18 weight parts of water, one weight part of the propolisextract of Example 1, two weight parts of citric acid, and appropriateamounts of a coloring and flavoring. The resultant mixture was formedinto gummy candies and then packed.

The product, which hardly causes carious teeth and exhibits a goodtexture and flavor, is useful as a health food to maintain or promotehealth.

EXAMPLE 8 Chewing Gum

To three weight parts of a gum base which had been heated to melt intosoftness, the following materials were added: four weight parts ofsucrose, three weight parts of maltose powders, 0.02 weight part of thepropolis extract of Example 2, and an appropriate amount of a coloring.The resultant mixture was kneaded and formed with a roller into chewinggum, which was then packed.

The product, which hardly causes carious teeth and exhibits a goodtexture and flavor, is useful as a health food to maintain or promotehealth.

EXAMPLE 9 Snack

Potatoes were stored to autodigest reducing sugars in the usual mannerat a temperature of 20° C. and a relative humidity of 85% for two weeks,then washed with water, peeled, selected, and sliced 1.5 mm thick with acentrifugal slicer. The slices were washed with water to remove thesurface starch, drained, fried with oil at 170° C. for about fiveminutes, and removed from oil. A seasoning powder was prepared by mixingsix weight parts of salt, three weight parts of food grade trehalosepowders "TREHAOSE®" (commercialized by Hayashibara Shoji, Inc., Okayama,Japan), and appropriate amounts of the propolis extract of Example 1 andspices, and the seasoning was added adequately to the slices with asalter. The seasoned slices were transferred to a weighing packer, wherethe slices were weighed, packed, and sealed. Thus a snack was obtained.

The product, with a good texture and flavor, is useful as a health foodto maintain or promote health.

EXAMPLE 10 Tea Bag

Nine weight parts of lyophilized black tea extract powders which hadbeen dissolved in an appropriate amount of water were mixed with oneweight part of the propolis extract of Example 2 which had beendissolved in an appropriate amount of ethanol. With the resultantmixture, ninety weight parts of black tea leaves were sprayed, afterbeing fermented and dehydrated in the usual manner. The leaves weresieved out, cut into pieces, dried for completion, separated fromimpurities with a separator, and divided and packed into two grams perbag of Japanese paper. Thus tea bags were obtained.

For use to drink, a bag of the product is soaked in about 180 ml of coldwater for ten minutes or in hot water (90° C. to 100° C.) for twominutes. The product, with good taste and flavor, is useful as a healthfood to maintain or promote health.

EXAMPLE 11 Custard Cream

The following materials were mixed in the usual the manner: 100 weightparts of corn starch, 40 weight parts of food grade trehalose powders"TREHAOSE®" (commercialized by Hayashibara Shoji, Inc., Okayama, Japan),60 weight parts of trehalose-containing syrup "TREHASTAR®"(commercialized by Hayashibara Shoji, Inc., Okayama, Japan), 80 weightparts of maltose, 20 weight parts of sucrose, and 0.25 weight parts ofthe propolis extract of Example 6, before being mixed with 280 weightparts of hen eggs. Into the resultant mixture 1,000 weight parts of milkwere moderately poured, and the mixture was stirred over fire till cornstarch was gelatinized so as to be wholly translucent. After the firewas stifled, an appropriate amount of a vanilla flavoring was added tothe mixture, which was then weighed, divided, and packed. Thus a custardcream was obtained.

The product, with mild luster and improved preservative activity, isuseful as a health food to maintain or promote health.

EXAMPLE 12 Supplemental Health Food

The following materials were mixed in the usual manner: 52 weight partsof food grade trehalose powders "TREHAOSE®" (commercialized byHayashibara Shoji, Inc., Okayama, Japan), 40 weight parts of cornstarch, 3.5 weight parts of propolis extract of Example 3, and 2.5weight parts of cellulose crystals. The resultant mixture was kneadedunder an appropriate amount of water spray and granulized in fluid. Thegranules were pulverized and spherized into material powders fortablets. The powders were admixed with two weight parts of sucrose fattyacid ester as a surfactant into homogeneity and processed by using atablet machine with a punch (11 mm in diameter) into tablets (about 300mg/tablet).

The product, which is easily ingestible and decayable in alimentarycanals, is also useful as a health food to keep or enhance health.

EXAMPLE 13 Hair Rinse

The following materials were mixed in the usual manner: one weight partof food grade trehalose powders "TREHAOSE®" (commercialized byHayashibara Shoji, Inc., Okayama, Japan), two weight parts of thepropolis extract of Example 1, two weight parts of α-glucosyl rutinpowders "αG Rutin" (commercialized by Toyo Sugar Refining Co., Ltd.,Tokyo, Japan), two weight parts of distearyl methylene ammoniumchloride, two weight parts of cetanol, two weight parts of silicon oil,one weight part of polyoxyethylene oleyl alcohol ether, and anappropriate amount of a flavoring. The mixture was heated into asolution, to which a mixture of three weight parts of 1,3-butyleneglycol, 85 weight parts of refined water, and an appropriate amount ofan antiseptic were added under stirring conditions. The resultantmixture was cooled, and thus a hair rinse was obtained.

The product, which is stable and does not irritate scalps, is alsouseful as a cosmetic to keep or promote healthy conditions of scalps andhair.

EXAMPLE 14 Milky Lotion

The following materials were mixed in the usual manner: 0.5 weight partof polyoxyethylene behenylether, one weight part of polyoxyethylenesorbitol tetraoleate, one weight part of lipophilic glycerolmonostearate, 0.5 weight part of pyruvic acid, 0.3 weight part ofbehenyl alcohol, 0.3 weight part of maltitol, one weight part of avocadooil, one weight part of the propolis extract of Example 3, andappropriate amounts of vitamin E and an antiseptic. The mixture washeated into a solution, to which one weight part of sodium L-lactate,seven weight parts of 1,3-butyleneglycol, 0.1 weight part ofcarboxyvinyl polymer, and 86.3 weight parts of refined water were added.The resultant mixture was emulsified with a homogenizer and mixed bystirring with an appropriate amount of a coloring. Thus a milky lotionwas obtained.

The product, which is less sticky and satisfactorily extendable, isuseful as a cosmetic to maintain or promote healthy conditions of skins.

EXAMPLE 15 Bath Liquid

Twenty-one weight parts of sodium DL-lactate, eight weight parts ofsodium pyruvate, five weight parts of the propolis extract of Example 2,40 weight parts of ethanol, 26 weight parts of refined water, andappropriate amounts of a coloring and flavoring were mixed in the usualmanner. Thus a bath liquid was obtained.

The product is used for bath in 1/100 to 1/10,000 dilution with warm orcool water. The product, with effectiveness of cleansing and whiteningskins and antimicrobial activity, is useful also as a cosmetic tomaintain or promote skin health.

EXAMPLE 16 Toothpaste

A toothpaste was prepared by mixing in the usual manner the followingmaterials: 45 weight parts of calcium secondary phosphate, 2.9 weightparts of pullulan, 1.5 weight parts of sodium lauryl sulfate, 20 weightparts of glycerine, 0.5 weight part of polyoxyethylene sorbitan laurate,10 weight parts of sorbitol, seven weight parts of maltitol, 13 weightparts of refined water, 0.1 weight part of the propolis extract ofExample 2, and an appropriate amount of a flavoring.

The product is stable and useful as a toiletry to maintain or promotehealthy conditions of mouths.

EXAMPLE 17 Ointment

The following materials were mixed in the usual manner: one weight partof sodium acetate trihydrate, four weight parts of calcium DL-lactate,and 10 weight parts of glycerine. To the resultant mixture, 0.5 weightpart of peppermint oil, 49 weight parts of petrolatum, 10 weight partsof Japan wax, 10 weight parts of lanolin, 14.5 weight parts of sesameoil, and one weight part of the propolis extract of Example 3 were addedand mixed into homogeneity. Thus an ointment was obtained.

The product, having a satisfactory permeability and extensibility, isuseful as a pharmaceutical to maintain or promote health conditions ofskins.

EXAMPLE 18 Feeding Fluid

In accordance with the formation in Table 5, the ingredients were mixed(in Table 5, "appropriate" amount corresponds to a half value of thedietary allowance for the life activity level II, moderate, in thereport "dietary allowances for Japanese" by Ministry of Health andWelfare of Japan):

                  TABLE 5                                                         ______________________________________                                                          Blended amount                                              Ingredient        (weight parts)                                              ______________________________________                                        powdered skim milk                                                                              43.0                                                        powdered whole milk                                                                             12.0                                                        trehalose *1      43.8                                                        the propolis extract                                                                             1.0                                                        of Example 3                                                                  vitamin A         appropriate                                                 vitamin D         appropriate                                                 thiamine hydrochloride                                                                          appropriate                                                 riboflavine       appropriate                                                 pyridoxine hydrochloride                                                                        appropriate                                                 cyanocobalamine   appropriate                                                 choline tartrate  appropriate                                                 nicotinamide      appropriate                                                 calcium pantothenate                                                                            appropriate                                                 2-O-a-D-glucopyranosyl-                                                                         appropriate                                                 L-ascorbic acid                                                               tocopherol acetate                                                                              appropriate                                                 iron sulfate      appropriate                                                 calcium hydrogenphospate                                                                        appropriate                                                 pullulan *2        0.2                                                        ______________________________________                                         *1: reagent grade trehalose powders (commercialized by Hayashibara            Seibutsu Kagaku Kenkyujo, Inc., Okayama, Japan)                               *2: pullulan powders with an averaged molecular weight of 150,000 daltons     ("PIF", commercialized by Hayashibara Shoji, Inc., Okayama, Japan)       

The resultant mixture was canned under sterile conditions in a contentof 500 g/can. Thus a product for a feeding fluid was obtained.

For use, a can of the product is dissolved in once or twice equalweights of cool or slightly warm (at 30° C. to 40° C.) water beforefeeding. In the case of feeding through peroral routs, the solution canbe applied by a single or several divisional shots per day; and throughtransnasal and gastric or interstitial fistula routs, by a continuous orintermittent flow at a flow rate of about 300 ml/hr. The product, whichsupplies calories, is useful as a pharmaceutical to maintain or promotehealth.

EXAMPLE 19 Wet Tissue Paper

A mixture was prepared with 0.5 weight part of the propolis extract ofExample 3, one weight part of succinic acid, 0.5 weight part ofglycerine, and 98 weight parts of refined water. With the mixture, 40weight parts of non-woven fabric of cuproxam cellulose fibers werewetted. Thus a wet tissue paper was obtained.

The product, which possesses germicidal and antimicrobial activity butis less irritating to skins, is useful as a daily life utensil tomaintain or promote health.

EXAMPLE 20 Antimicrobial Spray

A material liquid was prepared with 68 weight parts of isopropanol, 26weight parts of refined water, two weight parts of the propolis extractof Example 2, and four weight parts of benzoic acid, which werehomogenized in the usual manner. The material liquid was chargedtogether with liquefied carbonic acid gas as a propellant in 100ml-spray cans at a ratio of 40ml to 60 ml per can. Thus cans with anantimicrobial spray were obtained.

The product, which is safe and with a good germicidal and antimicrobialactivity, is useful as a daily life utensil to maintain or promotehealth.

As described above, this invention was made based on the identificationof the odorizing ingredients of propolis and the establishment of themethod to decrease the ingredients from propolis extracts withoutattenuating the inherent biological activities. The peculiar odor of thepropolis extract of this invention is apparently lower than that ofconventional propolis extracts. The present propolis extract gains avariety of uses in the fields that has rejected use of propolis becauseof its peculiar odor, such as the fields of foods, cosmetics,pharmaceuticals, and others that can use the biological activities ofpropolis. The present propolis extract can be produced in desiredamounts by the process of this invention comprising the steps ofextracting with a hydrophilic organic solvent and treating with ananion-exchange resin.

This invention exhibits these remarkable effects and greatly contributesto the art.

While there has been described what is at present considered to be thepreferred embodiments of this invention, it will be understood thevarious modifications may be made therein, and it is intended to coverin the appended claims all such modifications as fall within the truespirits of the invention.

We claim:
 1. A method for producing a propolis extract having a reducedcontent of at least one odorizing ingredient selected from odorizingingredients having a relative retention time against p-ethyl phenol of0.7 to 0.8 as determined by gas chromatography using p-ethyl phenol asan internal standard and odorizing ingredients having a relativeretention time against p-ethyl phenol of 1.2-1.3 as determined by gaschromatography, and mixtures thereof, comprising:soaking propolis in ahydrophilic organic solvent to effect extraction; and treating theliquid layer in the resultant extract with an anion exchange resin. 2.The process according to claim 1 wherein the hydrophilic organic solventis selected from the group consisting of methanol, ethanol, n-propanol,and isopropanol, optionally mixed with water.
 3. The process accordingto claim 1 wherein the hydrophilic organic solvent is ethanol.
 4. Theprocess according to claim 1 wherein the anion exchange resin is used incombination with a cation exchange resin.